1. Field of the Invention
The present invention relates to methods for producing polypeptides. The present invention also relates to isolated promoters and to nucleic acid constructs, vectors, and host cells comprising the promoters operably linked to nucleic acid sequences encoding polypeptides.
2. Description of the Related Art
The recombinant production of a heterologous protein in a fungal host cell, particularly a filamentous fungal cell such as Aspergillus, may provide for a more desirable vehicle for producing the protein in commercially relevant quantities.
Recombinant production of a heterologous protein is accomplished by constructing an expression cassette in which the DNA coding for the protein is placed under the expression control of a promoter, excised from a regulated gene, suitable for the host cell. The expression cassette is introduced into the host cell, usually by plasmid-mediated transformation. Production of the heterologous protein is then achieved by culturing the transformed host cell under inducing conditions necessary for the proper functioning of the promoter contained on the expression cassette.
The development of a new fungal host cell for the recombinant production of proteins generally requires the availability of promoters that are suitable for controlling the expression of the proteins in the host cell. Fusarium venenatum has been shown to be useful as a new host cell for such expression (WO 96/00787, WO 97/26330). Moreover, the promoter from the Fusariuin oxysporum trypsin-like protease gene has been described which is useful for expressing heterologous genes in Fusarium venenatum host cells (U.S. Pat. No. 5,837,847).
However, there is a need in the art for new promoters for controlling the expression of heterologous genes.
It is an object of the present invention to provide improved methods for producing a polypeptide in a fungal host cell and new promoters for such production.
The present invention relates to methods for producing a polypeptide, comprising: (a) cultivating a fungal host cell in a medium conducive for the production of the polypeptide, wherein the fungal host cell comprises a first nucleic acid sequence encoding the polypeptide operably linked to a second nucleic acid sequence comprising a promoter foreign to the nucleic acid sequence, wherein the promoter comprises a sequence selected from the group consisting of nucleotides 1 to 3949 of SEQ ID NO:1, nucleotides 1 to 938 of SEQ ID NO:2, and nucleotides 1 to 3060 of SEQ ID NO:3, and subsequences thereof; and mutant, hybrid, and tandem promoters thereof; and (b) isolating the polypeptide from the cultivation medium.
The present invention also relates to isolated promoters sequences and to constructs, vectors, and fungal host cells comprising one or more of the promoters operably linked to a nucleic acid sequence encoding a polypeptide.
The present invention also relates to methods for obtaining mutant promoters of nucleotides 1 to 3949 of SEQ ID NO:1, nucleotides 1 to 938 of SEQ ID NO:2, and nucleotides 1 to 3060 of SEQ ID NO:3.
The present invention also relates to isolated nucleic acid sequences, selected from the group consisting of:
(a) a nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 65% identity with amino acids 22 to 581 of SEQ ID NO:4, at least 65% identity with amino acids 19 to 200 of SEQ ID NO:5, or at least 65% identity with amino acids 1 to 187 of SEQ ID NO:6;
(b) a nucleic acid sequence having at least 65% homology with nucleotides 4013 to 5743 of SEQ ID NO: 1, at least 65% homology with nucleotides 993 to 1593 of SEQ ID NO:2, or at least 65% homology with nucleotides 3061 to 3678 of SEQ ID NO:3;
(c) a nucleic acid sequence which hybridizes under very low, low, medium, medium-high, high, or very high stringency conditions with (i) nucleotides 4013 to 5743 of SEQ ID NO:1, nucleotides 993 to 1593 of SEQ ID NO:2, or nucleotides 3061 to 3698 of SEQ ID NO:3; (ii) the cDNA sequence contained in nucleotides 4013 to 5743 of SEQ ID NO:1, nucleotides 993 to 1593 of SEQ ID NO:2, or nucleotides 3061 to 3698 of SEQ ID NO:3; (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii);
(d) a nucleic acid sequence encoding a variant of the polypeptide having an amino acid sequence of SEQ ID NO:4, SEQ ID. NO. 5, or SEQ ID NO:6 comprising a substitution, deletion, and/or insertion of one or more amino acids;
(e) an allelic variant of (a), (b), or (c); and
(f) a subsequence of (a), (b), (c), or (e).
The present invention further relates to constructs, vectors, and recombinant host cells of the nucleic-acid sequences.